Inhibition of angiogenesis

ABSTRACT

Angiogenesis in mammals is inhibited by administration of the active agents (1) heparin or a heparin fragment which is a hexasaccharide or larger or analogous compounds and (2) a steroid having 17α- and 21-hydroxy groups, 3- and 20-one groups, and in the 16-position hydrogen hydroxy or a methyl group, and non-toxic physiologically acceptable carboxylates, acetals, ketals and phosphates thereof.

This invention was made with U.S. Government support and the Governmenthas certain rights in the invention.

This application is a divisional of U.S. Ser. No. 07/080,255 filed July27, 1987, now abandoned which is a continuation of U.S. Ser. No.06/844,221 filed Mar. 24, 1986, now abandoned which is a continuation ofU.S. Ser. No. 06/641,305 filed Aug. 16, 1984 now abandoned which is acontinuation-in-part of U.S. Ser. No. 559,175 filed Dec. 7, 1983 whichis in turn a continuation-in-part of U.S. Ser. No. 451,431 filed Dec.20, 1982, now abandoned.

This invention relates to inhibition of angiogenesis and pertains morespecifically to treatment of mammals with heparin or heparin fragmentsor analogues and with steroids to inhibit angiogenesis with subsequentregression of large tumor masses and prevention of tumor metastasis inmammals containing such tumors.

Angiogenesis, the growth of new capillary blood vessels, is important innormal processes such as development of the embryo, formation of thecorpus luteum and wound healing. It is also a component in pathologicprocesses such as chronic inflammation, certain immune responses andneoplasia. Furthermore, angiogenesis is a property of most solid tumorsand is necessary for their continued growth.

It has previously been reported that heparin enhanced the intensity ofangiogenesis induced by tumors in vivo, although in the absence of tumorcells or tumor extract neither heparin nor the mast cells which releaseheparin could induce angiogenesis. Taylor and Folkman, Nature Vol. 297,307-312 (1982). It has also been reported in Shubik et al., J. Natl.Cancer Inst., Vol. 57, 769-774 (1976) that 6 α-methyl-prednisolonepartially suppressed tumor angiogenesis in hamster cheek pouch undercertain conditions, but tumor growth was not stopped, and many otherpublications have reported continued growth of tumors even in thepresence of large doses of cortisone. It has also been reported in Grosset al., Proc. Natl. Acad. Sci., U.S.A., Vol. 78, 1176-80 (1981) thatmedroxyprogesterone, dexamethasone, and to a lesser extent cortisone,inhibited tumor angiogenesis in rabbit corneas, while estradiol andtestosterone were ineffective

Heparin, an α,β glycosidically linked highly sulfated copolymer ofuronic acid and glucosamine, has been used clinically as ananticoagulant for half a century. Despite its importance and widespreaduse, both the exact structure of heparin and the precise nature by whichit acts in blood anticoagulation have not been elucidated. Much of thedifficulty in determining the structure of heparin is because it is nota homogeneous substance. Heparin is polydisperse with a molecular weightrange from 5,000 to 40,000. Within a given chain, there are alsostructural variations such as varying degrees of sulfation,N-acetylation, and C-5 epimerization in the uronic acid residue.

Consequently, the precise composition of commercial heparin variesdepending on its source and method of purification. Heparin has beendegraded by treatment with heparinase (an enzyme of bacterial origin,Langer et al. U.S. Pat. No. 4,341,869) which cleaves the molecule at theα-glycosidic linkages between N-sulfated-D-glucosamine 6-sulfate andL-iduronic acid 2-sulfate to form fragments including disaccharide,tetrasaccharide, hexasaccharide, and larger oligosaccharides, each beingsimply a chain-shortened heparin fragment with minor end groupmodification (the degradation results in a Δ-4,5 site of unsaturation inthe terminal uronic acid residue). Linhardt et al., J. Biol. Chem., Vol.257, 7310-13 (1982).

It has now been found that angiogenesis in mammals is inhibited andtumor masses in mammals are caused to regress (and metastasis prevented)by administration of the combination of two essential active agents: (1)heparin or a heparin fragment which is a hexasaccharide or larger, or ananalogous compound having one of the structures ##STR1## and (2) asteroid having 17α- and 21-hydroxy groups, 3- and 20-one groups, and inthe 16- position hydrogen hydroxy or a methyl group, and non-toxicphysiologically acceptable carboxylates, acetals, ketals and phosphatesthereof, said combination exhibiting a vascular zone when implanted inan immature chick chorioallantoic membrane.

Among the steroids which are effective are the following:

17α,21-dihydroxy-4-pregnene-3,11,20-trione and its 21-acetate (orcortisone)

11α, 17,21-trihydroxypregn-4-ene-3,20-dione (or 11α-hydrocortisone)

11β,17α,21-trihydroxypregn-4-ene-3,20-dione (or hydrocortisone)

17α,21-dihydroxypregna-4,9(11)-diene-3,20-dione

15α,17α,21-trihydroxy-4-pregnene-3,20-dione

16α,17α,21-trihydroxy-6α -methylpregn-4-ene-3,20-dione-2l-acetate-16,17cyclic ketal of acetone

6α-fluoro-17α,21-dihydroxy-16β-methylpregna-4,9(11)-diene-3,20-dione

6α-fluoro-17α,21-dihydroxy-16β-methylpregna-4,9(11)-diene-3,20-dione-17,2l-diacetate

6β,17α,21-trihydroxypregn-4-ene-3,20-dione

17α,21-dihydroxypregn-4-ene-3,20-dione-21-acetate

17α,21-dihydroxypregn-4-ene-3,20-dione

9β,11β-epoxy-17α,21-dihydroxy-2α-methylpregn-4-ene-3,20-dione-21-acetate

17α,21-dihydroxy-16α-methylpregn-4-ene-3, 20-dione

9α,11β-dichloro-17α,21-dihydroxypregn-4-ene-3,20-dione-2l-acetate

17α,21-dihydroxy-6α,16α-dimethylpregn-4-ene-3,20-dione-21-acetate

17α,21-dihydroxy-16α-methylpregna-4,9(11)-diene-3,20-dione-2l-acetate

17α,21-dihydroxy-16β-methylpregna-4,9(11)-diene-3,20-dione-2l-benzoate

17α,21-dihydroxy-6β-methylpregna-4,9(11)-diene-3,20-dione-21-acetate

6α-fluoro-17α,21-dihydroxy-16β-methylpregna-4,9(11)-diene-3,20-dione-l7-acetate-21-benzoate

17α,21-dihydroxy-16β-methylpregna-1,4,9(11)-triene-3,20-dione-17-succinate sodium monohydrate

9α-fluoro-11β,16α,17α,21-tetrahydroxypregn-4-ene-3,20-dione-16,21-diacetate

17α,21-dihydroxy-16α-methylpregna-1,4,9(11)-triene-3,20-dione-21-succinatesodium monohydrate

6α-fluoro-17α,21-dihydroxy-16β-methylpregna-1,4,9(11)-triene-3,20-dione-21-succinatesodium

More preferred are those steroids which lack glucocorticoid andmineralo-corticoid activity, since such activity is an undesired sideeffect and limits the dose size or extent of use of the steroid for thepurpose of the present invention. Among such more preferred steroids are11α, 17,21-trihydroxypregn-4-ene-3,20-dione (or 11α-hydrocortisone),17α,21-dihydroxypregn-4-ene-3,20-dione (or 11-desoxycortisol orCortexolone), and 17α,21-dihydroxypregna-4,9(11)-diene-3,20-dione.

It has also been found that certain steroids which display much greaterglucocorticoid activity than hydrocortisone, for example11β,17α,21-trihydroxy16α-methyl-9α-fluoropregna-1,4-diene-3,20-dione(also known as dexamethasone) and11β,17α,21-trihydroxypregna-1,4-diene-3,20-dione (also known asprednisolone) are inactive for the purposes of the present invention.

Neither mature non-growing blood vessels nor vascular tissue seems to beaffected by the procedure of the present invention. Inhibition ofangiogenesis in accordance with the present invention, in addition toits effect upon tumor regression and metastasis in tumor-bearingmammals, is effective as a contraceptive in females even if firstadministered after insemination has occurred, and is effective intreating diseases involving neovascularization such as neovasculardiseases of the eye.

None of the steroids themselves effectively inhibits angiogenesis norcauses regression of tumors in the absence of heparin or a heparinfragment or the specified compounds. Heparin alone does not inhibitangiogenesis but on the contrary enhances it.

The active agents may be mixed together prior to administration or maybe administered separately at about the same time so that both arepresent simultaneously in the mammal being treated. The administrationmay be oral or parenteral including inter alia topical application,intravenous, intra-arterial or subcutaneous injection, and includingabsorption as well as injection and introduction into bodily aperturesor orifices In the case of heparin, which is commercial available in theform of heparin sodium, oral administration leads to degradation in thegastrointestinal tract which results in loss of its anticoagulantactivity, but because it has been found that the degradation productsinclude disaccharide and larger fragments, this mode of administrationis highly effective for the present invention both for heparin forheparin fragments, and for the specified compounds. The heparin and itsfragments as well as the specified compounds may be employed in anyphysiologically acceptable non-toxic form, including their metal salts,preferably as the sodium salts, all of which are embraced in the term"heparin" or "fragment" or "compound" as used in the presentspecification and claims. For best results heparin sold under the tradename "Panheprin" (Abbott Laboratories) is preferred, but heparin fromother sources, such as Hepar, Inc. can also be used although lesseffective

Cortisone and its physiologically acceptable non-toxic derivatives suchas the acetate, as well as many other steroids useful in the presentinvention, are only very slightly soluble in water, hence are preferablyadministered parenterally, e.g. subcutaneously, not orally. For oraladministration, steroids such as hydrocortisone or its 11-α isomer(which are relatively water soluble as compared to cortisone) or one oftheir water-soluble physiologically acceptable non-toxic derivativessuch as a carboxylate, acetal, ketal or the phosphate are preferredWater-insoluble derivatives of water-soluble steroids such asderivatives of hydrocortisone or its 11- α isomer which are non-toxicand physiologically acceptable, are administered parenterally. The terms"cortisone" and "hydrocortisone" and 11- α isomer of hydrocortisone asused in the present specification and claims are intended to includeboth the steroids themselves and their derivatives as defined above.

Dosages employed are limited only by the well known limits for theadministration of the drugs individually for their usual effects, in thecase of cortisone, hydrocortisone, or its 11-α isomer. However, a numberof the useful steroids have no other biological effect apart from thepresent invention. Simple testing, for example, by the procedure of

Example 3 below, suffices to determine effectiveness and optimum dose.Heparin may be administered percutaneously in amounts as large astolerable without objectionable anticoagulant effects. Since heparinadministered orally has no anticoagulant effect, and since thehexasaccharide fragment has no anticoagulant effect whether given orallyor in any other way, large dosages can thus be administered without riskof bleeding. Oral dosages of heparin of the order of 27,000-45,000 unitsper kg. body weight per day have been found to be effective, but whenadministered subcutaneously, doses greater than about 600 units per kg.body weight twice daily led to undesirable anticoagulation effects. Inthe case of the hexasaccharide fragment, 7 mg per kg. body weight twicedaily subcutaneously has been found effective. Cortisone acetate waseffective in subcutaneous dosages of 250 mg/kg/day down to 37 mg/kg/dayand hydrocortisone was effective orally in amounts of 0.45 mg/mldrinking water (approximately 75 mg/kg/day). The 11- α isomer ofhydrocortisone is approximately equal to hydrocortisone in activity forthe purpose of the present invention.

The dose size required to bring about regression of tumors or to preventmetastasis varies to some extent depending upon the identity of thetumor, as does the length of time required to bring about completeregression of tumors. Tumor size at the beginning of treatment alsoaffects the length of time required for complete regression. Because ofthe occurrence of angiogenesis in psoriasis and arthritis, it isexpected that the present invention may be useful in treating thesediseases. Because administration of cortisone, with or without heparinor heparin fragments or the specified compounds, may result in pulmonaryinfection after a number of days, it is desirable to administer asuitable antibiotic as prophylaxis during treatment in accordance withthe present invention.

The active agents are best dissolved or suspended in a suitable carrierwhich itself must be non-toxic and physiologically acceptable, such aswater or normal saline. Compositions containing mixtures of the activeagents, either dry or in a suitable carrier, can be employed.

EXAMPLE 1

Cortisone acetate 0.9 mg in 0.9 ml saline was flooded over thechorioallantoic membrane of 8-day chick embryos through a window in theshell made previously. On day 9, tumor extract (100 μg) from hepatomacells (as described in Zetter, Nature Vol. 285, 41-43, 1980) in 5 μl H₂O was placed on the center of a round 15 mm diameter plastic coverslipand allowed to dry. To the center of each coverslip was then added a 5μl aliquot containing either heparin (6 μg, i.e., 1 unit), or water, atleast twenty embryos being subjected to each. After drying, thecoverslip was placed on the chorioallantoic membrane: Additional controlembryos received tumor extract and/or heparin, but were not pre-treatedwith cortisone acetate. The membranes were viewed on day 11 with a ×12stereoscope. Angiogenesis was present if new capillaries were seen toconverge on the spot in the center of the coverslip. All of the embryostreated with water or heparin but no cortisone exhibited angiogenesis,as well as 80% of those treated with cortisone acetate alone. Less than2% of those treated with both heparin and cortisone acetate exhibitedangiogenesis.

EXAMPLE 2

Porcine mucosal heparin was exhaustively degraded using heparinase bythe procedure of Langer et al , Science Vol. 217, 261-3 (1982) and theproducts were fractionated using Sephadex columns equilibrated with 1MNH₄ OAc. The degraded heparin had no anticoagulant activity asdetermined by activated partial thromboplastic time or whole bloodrecalcification time. The product or product mixture (250 mg) wasdissolved in 1 cc of 1M NH₄ OAc, loaded onto a 75×2.5 cm G-15 column,and eluted at 0.5 cc/min. This resulted in several incompletely resolvedpeaks corresponding to tetra-, hexa-, and higher oligosaccharides and aseparate peak corresponding to disaccharide product. The disaccharidepeak was freeze-dried, dissolved in 0.2 cc of 1M NH₄ OAc andrechromatographed on G-15 resulting in the same sharp peak which wasfreeze-dried. The mixture of tetra-, hexa-, and oligosaccharides wasfreeze-dried, redissolved in 1 cc of 1M NH OAc, and eluted from a50×1.25 cm G-50 at 2 cc/min, resulting in an unresolved double peakcorresponding to tetra- and hexasaccharide fragments and an additionalpeak corresponding to oligosaccharides which was freeze-dried. Thetetra- and hexasaccharide fragments were combined, freeze-dried,redissolved in 1 cc of 1M NH₄ OAc, and loaded onto a G-15 column. Thetetrasaccharide was eluted from a G-15 column in a single peak, thecenter cut of which was freeze-dried. The hexasaccharide fraction wasfreeze-dried, redissolved in 0.3 cc 1M NH₄ OAc, eluted from a G-15column in a single peak, the center cut of which was freeze-dried.

Fragment size was determined by dissolving a weighed amount of eachfraction into 0.03M hydrochloric acid and measuring the absorbance ofthis solution at 232 nm. The molecular weight of each fragment wascalculated using a molar absorptivity, for the α, β unsaturatedcarboxylate end group present in each of these products, of ξ=5500. Thedi- and tetrasaccharides were further characterized by comparing their Kavg-values on G-15 with mono-, di-, and trisaccharide standards.Measured molecular weights were 530, 1210, 1600, and 1870 for the di-,tetra-, hexa-, and oligosaccharide fractions respectively.

The various heparin fragments were dissolved into methylcellulose discseither alone or with cortisone acetate. The discs were then applied tothe 4-day yolk sac membrane of chick embryos cultured in Petri dishes asdescribed by Taylor and Folkman, Nature, Vol. 297, 307-312 (1982). Inthe presence of cortisone acetate (100 μg), as shown in the followingtable, the hexasaccharide fragment demonstrated the highestantiangiogenesis activity.

                  TABLE I                                                         ______________________________________                                        Percent Embryos That Developed Avascular Zones 48 Hours                       After Implantation of Methylcellulose Discs                                   On The 4-day Old Yolk Sac Membrane                                                    OLIGO-     HEXA-     TETRA   DI-                                      CONC.   SAC-       SAC-      SAC-    SAC-                                     (μg) CHARIDES   CHARIDE   CHARIDE CHARIDE                                  ______________________________________                                        12      all died   100%      0%      0%                                       8       100%       100%                                                       4       25%        100%                                                       1       25%         50%                                                       0.1       0         50%                                                       ______________________________________                                    

Tetra- and disaccharides were inactive. Oligosaccharides were lessactive and were toxic at higher concentrations. Therefore, thehexasaccharide fragment was used in subsequent experiments. In thegrowing 6-day chorioallantoic membrane, discs containing hexasaccharide(12 μg) and cortisone acetate (100 μg) produced large avascular zones upto 12.6±0.1 mm diameter by 48 hours. As in the case of heparin (withcortisone acetate), capillaries in the mesodermal layer were absentwhile the other two tissue layers of the membrane were intact andviable. Hexasaccharide alone did not promote tumor angiogenesis asheparin did.

All discs contained a combination of cortisone acetate (100 μg) and aheparin fragment. No avascular zones developed in the presence of anyheparin fragment alone, or with cortisone or methylcellulose alone. Tenembryos were used for each group. With hexasaccharide (plus cortisone),the area of the avascular zone was 17% of the vascular membrane at 12 μgand 15% at 0.1 μg. For the oligosaccharides, the maximum avascular areawas 10%.

EXAMPLE 3

Fertilized chick embryos were removed from their shell on day 3 (or 4)and incubated in a Petri dish in high humidity and 5% CO₂ as previouslydescribed by Auerbach et al., J. Devel. Biol., Vol. 41, 391-4 (1974),except that an outer dish and antibiotics were not used. On day 6, amethylcellulose disc (10 μl) containing either heparin (6 μg), orhexasaccharide heparin fragment (12 μg), or cortisone acetate (Sigma,powder free of preservatives and suspending agents), or a combination ofcortisone acetate+heparin or cortisone acetate+hexasaccharide wasimplanted on the chorioallantoic membrane. The embryos were examined 48hours later, and if a clear avascular zone appeared around themethylcellulose disc, the diameter of the zone was measured with a NikonProfile projector at ×20. Thirty embryos were used in each group. Indiaink was injected into the heart of some embryos just before formalinfixation so that vessels could be followed to the edge of the avascularzone in histological sections.

Hexasaccharide+cortisone acetate produced avascular zones of 12.6±0.1 mmdiameter in all embryos. Heparin+cortisone acetate produced avascularzones of 8.9±0.7 mm diameter. There were no avascular zones in thepresence of any compounds alone, or with methylcellulose alone.

Histologic cross-sections of the chorioallantoic membranes, revealedthat capillaries developed normally in the presence of any compoundalone. In contrast, capillaries were completely absent from themesodermal layer in the face of either hexasaccharide+cortisone acetate,or heparin+cortisone acetate, while the ectodermal and endodermal celllayers remained unaffected. In the mature chorioallantoic membrane wherevessels are no longer growing, the cortisone acetate-heparin or-hexasaccharide fragment combinations were without effect.

Avascular zones were also observed using essentially the same procedurewith a combination of heparin (Panheprin) (10 units) with 25-200 μg ofeach of the following steroids:

11β, 17α, 21-trihydroxypregn-4-ene-3,20-dione

11α, 17α, 21-trihydroxypregn-4-ene-3,20-dione

17α,21-dihydroxypregn-4-ene-3,20-dione

17α,21-dihydroxypregna-4,9(ll)-diene-3,20-dione

9α-fluoro-11α,16α,17α,21-tetrahydroxypregna-1,4-diene-3,20-dione (ortriamcinolone)

9α,11β-dichloro-17α,21-dihydroxypregn-4-ene-3,20-dione-21-acetate

17α,21-dihydroxy-16β-methylpregna-4,9(11)-diene-3,20-dione-21-benzoate

17α,21-dihydroxy-16β-methylpregna-1,4,9(11)-triene-3,20-dione-21-succinatesodium monohydrate

6α-fluoro-17α,21-dihydroxy-16β-methylpregna-4,9(11)-diene-3,20-dione

6α-fluoro-17α,21-dihydroxy-16β-methylpregna-4,9(11)-diene-3,20-dione-17,21diacetate

15α,17α,21-trihydroxy-4-pregnene-3,20-dione

16α,17α,21-trihydroxy-6α-methylpregn-4-ene-3,20-dione-21-acetate-16,17-cyclicketal of acetone

Of the foregoing, the last four displayed the greatest anti-angiogenicactivity by this test.

All of the effective combinations of active agents, for the purposes ofthe present invention, exhibit an avascular zone when implanted on animmature chick chorioallantoic membrane as described in this Example 3.

EXAMPLE 4

Polymer pellets of ethylene vinyl acetate copolymer (EVA) ofapproximately 1 mm diameter were impregnated, using the procedure ofLanger et al., Nature, Vol. 263, 797-800 (1976), with heparin 180 μg(Sigma), or hexasaccharide fragment 300 μg, or cortisone acetate 1.5 mg(Sigma), or a combination of cortisone and heparin. The pellets wereimplanted in the cornea of a rabbit eye 1 mm from the limbus and a 1 mm³piece of V2 carcinoma was implanted distal to the polymer, 2 mm from thelimbus. In the opposite eye of each rabbit, control pellets that wereempty were similarly implanted in juxtaposition to the tumor.

Release rates averaged 15 μg/day for heparin; 21 μg/day forhexasaccharide fragment; and 5 μg/day for cortisone. When the compoundswere mixed, they released at the same rates. By spectrophotometry, thepellets released heparin for 14 days, hexasaccharide for 11 days, andcortisone for more than 30 days.

As capillary blood vessels grew towards the tumor implant, maximumvessel length was measured every 3 days with a stereoscopic slit lamp at×10 (±0.1 mm). On day 14 the rabbits were killed, and India ink wasinjected into each carotid artery. The corneas were removed and examinedwith a stereoscope.

New capillary blood vessels were observed growing towards the tumor andpassing over an empty pellet or a pellet containing heparin alone at amean rate of 0.44 mm/day; and over a pellet containing cortisone aloneat 0.22 mm/day. The tumors behind these pellets were vascularized by 6-8days. When the pellets contained both cortisone and heparin, there wasno capillary growth for 13 days. When the heparin-cortisone pellets wereremoved or when the pellets were depleted of heparin, capillary growthresumed. Histologic sections showed that tumor cells remained viable andcapable of replication even when they were adjacent to theheparin-cortisone pellet.

In the presence of implanted pellets in which the hexasaccharidefragment of Example 2 replaced the heparin, new capillaries grew towardthe tumors at a mean rate of 0.30 mm/day in the presence of thehexasaccharide pellets; 0.14 mm/day when the pellets containedcortisone; and 0.32 mm/day when the pellets were empty. In the presenceof the hexasaccharide-cortisone combination, there was no capillarygrowth throughout the 13-day observation period in 4 rabbits, and in onerabbit a few capillaries grew at 0.07 mm/day.

EXAMPLE 5

(a) Reticulum cell Sarcoma: 1 mm³ pieces of tumor were implanted with atrocar subcutaneously in the backs of 30 mice; 5 per group. Treatmentwas begun 10 days later, when mean tumor volume was 1.5×10² mm³. Oralheparin was 200 U/ml in drinking water, average daily consumption was5-10 ml water per 22 g mouse. Cortisone acetate was administeredsubcutaneously once daily in a dose of 250 mg/kg for six days, 125 mg/kgfor one day, 75 mg/kg for one day, and thereafter a daily maintenancedose of 37 mg/kg (a "tapered" dosage). Control animals received eithersaline injections, or heparin alone or cortisone acetate alone. Allcontrols were dead by day 34 with large primary tumors and lungmetastases. All mice treated with oral heparin+cortisone tapered dosagebecame tumor-free by day 15 and remained so after treatment wasdiscontinued.

An additional group of mice was treated similarly except that heparinwas administered twice daily subcutaneously in a dose of 627 units, andcortisone acetate was administered subcutaneously once daily in auniform dose of 75 mg/kg. Response of the mice was the same as in thefirst set except that tumors recurred after cessation of treatment;these mice became permanently tumor-free when subjected to the regimenof oral heparin and cortisone acetate tapered dosage described above.One of these mice died on day 31 with no gross primary tumor and nometastases.

(b) Lewis Lung Carcinoma: Treatment began 7 days after implantation of a1 mm³ piece of tumor in 42 mice: 7 per group. Treatment was with oralheparin and subcutaneous cortisone acetate tapered dosages describedabove. All controls died by day 33 with large tumors and numerous lungmetastases. In the heparin+cortisone acetate groups treatment wasdiscontinued for each mouse after tumor had been invisible forapproximately 7 days. In the oral heparin+cortisone acetate all micewere off treatment by day 33, and remained tumor-free. In an additionalgroup treated with subcutaneous heparin and subcutaneous cortisoneacetate (75 mg/kg), 5 mice were off treatment at day 37 and remainedtumor-free. Two mice died of pneumonia on days 30 and 33 respectivelywith small primary tumors (<75 mm³). One metastasis was found in onemouse.

To determine if other steroids could substitute for cortisone, heparinwas administered with hydrocortisone, dexamethasone, ormedroxyprogesterone. Only hydrocortisone was as effective as cortisoneacetate in causing tumor regression when administered with heparin. Atthe highest tolerable doses neither dexamethasone (3.2 mg/Kg), normedroxyprogesterone (112 mg/Kg), caused regression of Lewis lung tumorswith or without heparin.

(c) B-16 Melanoma: 7.4×10⁶ melanoma cells were injected subcutaneouslyinto 40 mice; 5 per group. Treatment of one group was with oral heparinas described above and oral hydrocortisone, 0.45 mg/ml in drinking waterAnother group received oral heparin and subcutaneous cortisone acetatetapered dosage, and a third group received subcutaneous heparin andcortisone acetate 75 mg/kg. Controls received either water, or heparinalone, or hydrocortisone or cortisone acetate alone. All control animalsdied by day 31 with large tumors and lung metastases. In theheparin+cortisone acetate groups, treatment was discontinued after tumorhad become invisible for approximately 7 days. In the oralheparin+cortisone acetate tapered dosage group, 1 mouse died on day 24with a partially regressed tumor and 2 lung metastases that wereavascular and measured less than 0.1 mm. All other mice in the groupbecame tumor-free and remained so after their treatment was discontinuedby day 32. In the subcutaneous heparin+cortisone acetate group one mousedied on day 18 and another on day 21; neither had lung metastases.Treatment was discontinued for the other 3 mice on day 32; tumors 3weeks later were successfully re-treated with oral heparin+cortisoneacetate tapered dosage, and these mice have remained tumor-free In theoral heparin+oral hydrocortisone group, all mice remained tumor-freeafter their treatment was discontinued on day 47. The regimen of oralheparin+oral hydrocortisone seemed to be more effective for melanomathan it was for ovarian sarcoma or Lewis lung carcinoma.

(d) Bladder Carcinoma: 70 mice: 7 per group received a 1 mm³ implant oftumor subcutaneously. All control animals died by day 31, with largeprimary tumors. No mice bearing bladder carcinoma developed lungmetastases. One group was treated with oral heparin and subcutaneouscortisone acetate tapered dosage, starting on day 9 when mean tumorvolume was 140 mm³. Tumors stopped growing, but regressed onlypartially, and then reached a steady state where tumor volume remainedat approximately 70 mm³ for as long as the treatment was continued(i.e., 61 more days). Only one mouse died of pneumonia on day 19. The"dormant" tumors were viable, as evidenced by resumption of tumor growthwhenever treatment was discontinued for one mouse at a time, beginningat day 70. For a second group treated with subcutaneous heparin andsubcutaneous cortisone acetate 75 mg/kg as described above, the resultwas similar; i.e., long-term tumor "dormancy". One mouse died at day 21.

Because of the inability the standard regimen of oral heparin+cortisoneacetate to bring about complete regression, higher concentrations oforal heparin were used with other groups. With oral heparin (600U/ml)-cortisone acetate tapered dosage, there was more significant tumorregression and a steady state ("dormancy") was reached at a smallertumor volume of approximately 45 mm³. One mouse died. However, with 1000U/ml heparin, there was complete regression; mice remained tumor-freeafter discontinuation of treatment on ay 39. No mice died in this group.

In summary, all tumors either stopped growing or regressed when theheparin-cortisone acetate combination was administered. In contrast,when either compound was used alone, tumor growth continued at the samerate as in animals receiving only saline injections; all such controlanimals died with a large tumor burden.

In the majority of animals treated with heparin+cortisone acetate, itwas possible to achieve "complete regression", i.e., tumors did notrecur after treatment was discontinued. Thus, with the most effectiveregimen, oral heparin (200 U/ml)+cortisone (s.c. 250-tapered dosage), itwas possible to obtain "complete regression" in 100% of ovariansarcomas, 100% of Lewis lung carcinomas and 80% of B-16 melanomas.However, when bladder carcinomas were treated with this regimen, therewere no "complete regressions" until heparin was increased to 1000 U/ml,following which 100% of tumors regressed without recurrence. With theless effective regimen, heparin (s.c.)+cortisone (75 mgm), completeregression rate was ovarian, 80%; Lewis lung, 71%; B-16 melanoma 60%;and bladder 0%.

EXAMPLE 6

The hexasaccharide heparin fragment of Example 2 was dissolved insaline, 1.5 mg/ml. Three mice bearing implanted ovarian sarcoma weretreated with subcutaneous injection of the hexasaccharide fragment twicedaily at a dosage of 7 mg/kg and subcutaneous injection of cortisoneacetate- tapered dosage. Control mice received either cortisone acetatealone or saline. While control tumors grew progressively, thehexasaccharide+cortisone acetate treated tumors regressed rapidly andwere barely visible 4 days later. Their treatment with hexasaccharidewas then discontinued, and the tumors reappeared 3-5 days later.

EXAMPLE 7

To directly observe avascular tumors during systemic therapy, Lewis lungtumors were implanted in the mouse cornea by the procedure ofMuthukkaruppan et al., Science, Vol. 205, 1416-18 (1979) and treatmentwas begun 24 hours later. Heparin (oral)-cortisone acetate tapereddosage significantly inhibited capillary growth (0.02 mm/day) comparedto cortisone acetate alone (0.24 mm/day), heparin alone (0.32 mm/day) orsaline (0.23 mm/day). In the presence of heparin-cortisone acetate, athin plate of tumor remained avascular. Three-dimensional tumor-growthdid not occur. In contrast, in the saline controls or when eitherheparin or cortisone acetate were administered alone, tumors becamevascularized and grew as a three-dimensional mass until they eventuallyperforated the cornea. These large tumors could be regressed to theflat, thin intracorneal phase by the resumption of the heparin-cortisoneacetate combination. However, the intracorneal tumor cells could not beeradicated; discontinuation of the heparin-cortisone acetate led torecurrence of a vascularized tumor.

Lung metastases were counted in all animals that died. A ×6 stereoscopewas used. In all control animals, the lungs were heavily studded withmetastases from the three types of metastasizing tumors. In contrast,when any combination of heparin+cortisone acetate was used, nometastases were found in mice bearing ovarian sarcoma; only 1 metastasiswas found in a mouse bearing Lewis lung carcinoma; and 2 avascularmetastases less than 0.1 mm diameter were found in one mouse bearingB-16 melanoma. The nearly complete absence of metastases inheparin+cortisone treated mice was so striking, that the effect can bemore readily appreciated by the following expression of data

Total Number Lung Metastases

    ______________________________________                                         Total Number Lung Metastases                                                 ______________________________________                                        Controls =            4553 in 73 animals                                      Heparin + Cortisone =                                                                              3 in 39 animals                                          ______________________________________                                    

Furthermore, no lung metastases appeared in any surviving animals thatwere off treatment.

To exclude the possibility that tumor regression might be caused bydirect cytotoxicity, all 4 types of tumor cells were cultured in thepresence of 10% serum obtained from mice receiving either heparin,cortisone acetate, heparin-cortisone acetate, or no drug.Heparin-cortisone acetate did not inhibit cell growth, but in factstimulated it. Furthermore, histological sections shows no evidence of acytotoxic effect on bone marrow or intestinal mucosa in animalsreceiving heparin-cortisone acetate.

To exclude the possibility that heparin-cortisone acetate might inducetumor regression by promoting an immune reaction, mice were inoculatedwith fresh tumor cells at various intervals after they were offtreatment. These tumors grew at the same rate as the original implants.Furthermore, if tumor regression was nearly complete andheparin-cortisone acetate was discontinued prematurely, the originaltumor resumed its growth. Finally, by stopping and starting treatment,or by using sub-optimal doses of heparin-cortisone, bladder tumors couldbe maintained at nearly a constant small size (i.e., 45 to 70 mm³) forperiods of more than 8 weeks.

Inflammatory angiogenesis induced by implantation of silica particlesinto the rabbit cornea, immune angiogenesis induced by implantation oflymph node from a different rabbit, were also completely prevented bythe cortisone-heparin pellets. Cortisone by itself temporarily delayedthe onset of both types of angiogenesis (compared to an empty pellet),and heparin by itself delayed the onset of immune angiogenesis, butneither alone prevented angiogenesis for an extended period of time asdid the cortisone-heparin combination.

EXAMPLE 8

A human colon carcinoma was inoculated subcutaneously into nude[athymic] mice, and allowed to grow to a volume of 0.5 cm³. Controls andtreated animals received the same compounds, except that heparin in thedrinking water was 1000 U/ml. also an additional treatment groupincluded oral hydrocortisone (0.45 mg/ml) and oral heparin. Animals werehoused in cages protected by millipore filter. Tumors grew progressivelyin all control animals, but regressed in animals treated with heparinand cortisone or heparin and hydrocortisone. The treated tumors werebarely palpable after 6 weeks of therapy.

EXAMPLE 9

CD-1 "Swiss" mice were used because this strain breeds easily and thefertilized females almost always conceive and subsequently deliver afull litter.

One male was left alone in the cage for 24 hours. His bedding was thenchanged and two females were added at 5 p.m. The females were thenchecked at 8 a.m. the next morning for the presence of a cervical plug,which indicates insemination. Sufficient male-female cages were set upto obtain at least 20 pregnant females.

Treatment of the inseminated females was started on the day afterinsemination by offering drinking water to each of four groups, asfollows:

    ______________________________________                                        Group I        Heparin (Hepar) 1,000 U/ml                                                    in water                                                       II             Hydrocortisone phosphate,                                                     0.45 mg/ml in water                                            III            (I) and (II) together in water                                 IV             Water alone                                                    ______________________________________                                    

The treatments were continued for only four days, after which femalemice were placed one per cage, and followed closely for the presence ofoffspring. The cages were checked daily for any sign of abortion (fur,fetal remains, etc.).

Groups I, II and IV all produced healthy litters. In Group III therewere no mice born and no evidence of abortion. This supports theconclusion that anti-angiogenesis by heparin-cortisone inhibitsimplantation, presumably by inhibiting capillary growth from the uterus.

EXAMPLE 10 Inhibition of Tumor Vessels in the Rabbit Cornea

17α,2l-Dihydroxypregna-4,9(11)-diene-3,20-dione (ordelta-9(11)-deoxycortisol) (90 mg/ml) and Sigma heparin (1.8 mg/ml) weredissolved in 2 cc of 10% ethylene vinyl acetate (EVA) copolymer. A 1-2mm pellet was cut from the polymer and implanted into the pocket of arabbit cornea 1 mm from the limbus. The polymer was positioned betweenthe limbal vessels and a 1 mm³ piece of V2 Carcinoma implanted distal tothe pellet. The opposite eye of each of the 3 rabbits tested containedempty polymers. Mean vessel growth or 0.45 mm/day over a period of 16days was seen with the control corneas and 0.07 mm/day was seen in theeyes containing steroid and heparin Measurements are expressed as themean±SD. Similar results have been demonstrated with cortexolone andheparin implanted in the same manner.

What is claimed is:
 1. A composition for treating mammals for stoppinggrowth, bringing about regression, or preventing metastasis of solidtumors in mammals by oral, or parenteral administration in which theactive agents consist essentially of effective proportions for saidtreating of (1) a member selected from the group consisting of asaidcomposition including a non-toxic physiologically acceptable carrieradapted for oral or parenteral administration, said compositionexhibiting an avascular zone when implanted in an immature chickchorioallantoic membrane. heparin fragment which is either ahexasaccharide or larger oligosaccharide and an analogous compoundhaving of the structures ##STR2## and (2) a member selected from thegroup consisting of steroids having a 17α- and 21-hydroxy groups, 3- and20-one groups, and in the 16-position hydrogen, hydroxy, or a methylgroup, and a non-toxic physiologically acceptable carboxylate, oracetal, or ketal, or phosphate thereof.
 2. A composition as claimed inclaim 1 in which the active agents consist essentially of a heparinfragment which is either a hexasaccharide or larger oligosaccharide andcortisone.
 3. A composition as claimed in claim 1 in which the activeagents consist essentially of a heparin fragment which is eitherhexasaccharide or larger oligosaccharide and hydrocortisone.
 4. Acomposition for treating mammals for stopping growth, bringing aboutregression, or preventing metastasis of solid tumors in mammals by oraladministration only, in which the active agents consist essentially ofeffective proportions for said treating of (1) a member selected fromthe group consisting of heparin and a heparin fragment which is either ahexasaccharide or larger oligosaccharide and an analogous compoundhaving one of the structures ##STR3## and (2) a member selected from thegroup consisting of steroids having 17α- and 21-hydroxy groups, 3- andin the 16-position hydrogen, hydroxy, or a methyl group, and a non-toxicphysiologically acceptable carboxylate, or acetal, or ketal, orphosphate thereof, said composition including a non-toxicphysiologically acceptable carrier adapted for oral administration only.said composition exhibiting an avascular zone when implanted in animmature chick chorioallantoic membrane.
 5. The composition as claimedin claim 4 in which the active agents consist essentially of (1) heparinand (2) cortisone.
 6. The composition as claimed in claim 4 in which theactive agents consist essentially of (1) heparin and (2) hydrocortisone.7. A method of treating mammals bearing solid tumor masses to stopgrowth, to bring about regression or to prevent metastasis thereof whichcomprises administering orally thereto a dose effective for saidtreating of active agents consisting essentially of (1) heparin or aheparin fragment which is a hexasaccharide or larger oligosaccharide oran analogous compound having one of the structures ##STR4## and (2) asteroid having 17 α- and 21-hydroxy groups, 3- and 20-one groups, and inthe 16- position hydrogen, hydroxy, or a methyl group, and a non-toxicphysiologically acceptable carboxylate, or acetal, or ketal, orphosphate thereof, wherein said tumor masses are susceptible to saidtreatment with said active agents.
 8. The method as claimed in claim 7in which administration is oral and the active agents consistsessentially of heparin and hydrocortisone.
 9. The method as claimed inclaim 7 in which administration is oral and the active agents consistessentially of a hexasaccharide heparin fragment and hydrocortisone. 10.The method as claimed in claim 7 in which said administration is oraland said active agent (1) is heparin.
 11. A method of treating mammalsbearing solid tumor masses to stop growth, to bring about regression orto prevent metastasis thereof which comprises administering thereto aneffective dose effective for said treating of active agents consistingessentially of (1) a heparin fragment which is either a hexasaccharideor larger oligosaccharide or an analogous compound having one of thestructures ##STR5## and (2) a steroid having 17α- and 21-hydroxy groups,3- and 20-one groups, and in the 16- position hydrogen, hydroxy, or amethyl group, and a non-toxic physiologically acceptable carboxylate, oracetal, or ketal, or phosphate thereof, wherein said tumor masses aresusceptible to said treatment with said active agents.
 12. The method asclaimed in claim 11 in which the active agents consist essentially of(1) either said heparin fragment or analogous compound and (2)cortisone.
 13. The method as claimed in claim 11 in which the activeagents consist essentially of (1) said heparin fragment and (2)cortisone.
 14. The method as claimed in claim 11 in which the activeagents consists essentially of (1) either said heparin fragment oranalogous compound and (2) hydrocortisone.
 15. The method as claimed inclaim 11 in which said active agents consist essentially of (1) saidheparin fragment and (2) hydrocortisone.